Antigen-antibody reactions are part of the naturally occurring immune response of man and other animals upon exposure to foreign substances. Antibodies are derived from a class of proteins found in blood serum referred to as immunoglobulins. The presence of antigenic substance in the host organism results in the synthesis of immunoglobulin capable of binding to the antigen to form an antibody-antigen complex. The binding is characterized by a high degree of specificity and a high association constant between the antibody and antigen. The region of the antigen which participates in binding is called the epitope, and the region of the immunoglobulin participating in binding is called paratope. The high degree of specificity and affinity between antibodies and antigens has permitted the development of highly sensitive and specific methods of determining the presence and/or concentration of these substances. The methods are significant in the diagnosis of disease. Detection of antigens or antibodies to antigenic substances depends on the ability to distinguish between antibody-antigen complexes and the free forms of either antigen or antibody.
There are a number of prior art techniques for detection of antigens and antibodies. These methods include agglutination reactions with cells to form an insoluble particulate of latex or silica, gel diffusion, complement fixation, immunoelectrophoresis, Western blotting, fluorescent antibody techniques, radioimmunoassay, sandwich enzyme-linked immunoabsorbent assays and enzyme modulation immunoassays.
Immunochemical tests have important application for detecting the human immune deficiency virus (HIV) associated with the disease acquired immune deficiency syndrome (AIDS). The usefulness of determining the presence of the HIV in blood or blood products is evidenced by the resulting reduction in the number of persons contracting the disorder AIDS by receiving HIV in contaminated blood or blood products. Methods for the detection of antibodies to HIV and antigens produced by HIV have been described in the literature. (See, e.g., Groopman et al., Serological Characterization of HTLV-III Infection in AIDS and Related Disorders, "153 J. of Infectious Diseases 736 (1986); Allen, "A New HTLV-III/LAV Encoded Antigen Detected by Antibodies from AIDS Patients," 230 Science 810 (1985); Sarngadharan et al., "Antibodies Reactive with Human T-Lymphophilic Retrovirus (HTLV-III) in the Serum of Patients with AIDS," 224 Science 506 (1984); Gallo et al., "Frequent Detection and Isolation of Cyclopathic Retrovirus (HTLV-III) from Patients with AIDS and at Risk for AIDS," 224 Science 500 (1984).
One test currently employed for the detection of HIV is based on a technique called ELISA (enzyme-linked immunoabsorbent assay). According to this method, viral proteins from disrupted viral particles are coated on a plastic surface. The protein is exposed to a patient's serum and allowed to incubate. If the patient's serum contains antibodies to these proteins, they will bind to the viral proteins on the plastic surface. Unbound antibodies are removed by washing. Anti-human antibodies labelled with an enzyme (signal amplifier) are then incubated with the washed plastic surface. The enzyme-labelled antibodies will bind to any HIV antibodies retained on the HIV-coated plastic surface. Then a second wash is performed to remove any unbound anti-human antibodies. A substrate is added, which is converted by the enzyme label to a colored product indicating the presence of HIV antibodies in the sample. Although the method has good sensitivity and specificity, any positive specimen must be confirmed by Western blotting. This HIV test method requires expensive equipment, employs a time-consuming protocol and requires highly skilled personnel to perform the procedure. The technique is not easily performed by physicians in their offices, but requires the collection of a specimen for testing to be performed in a laboratory. Shipping of the specimen may lead to loss, damage or deterioration of the specimen prior to completion of the testing.
The presence of detectable antigen and antibodies to HIV during the course of the AIDS infection is not predictable with certainty and varies among patients. Generally, HIV antigen can be detected throughout the course of the infection, although there have been exceptional cases where neither antigen nor antibodies has appeared until 14 months after infection. IgM antibodies to HIV can usually be detected 2-3 weeks following exposure, and IgG antibodies appear detectably by the 12th week. Current FDA-approved AIDS tests detect only IgG antibodies, which means that there is a period during which infected blood may slip through screening procedures. Like ELISA, the present invention offers the advantage of being able to detect both IgM and IgG antibodies to HIV. In addition, IgA and IgE antibodies to HIV can also be detected. Thus, the period during which antibodies to HIV cannot be detected in blood specimens is considerably reduced by employing the immunoassay of the present invention.
Therefore, the object of the present invention is to provide an immunochemical assay for the determination of the presence or concentration of antigen or antibody in a fluid sample that is highly sensitive and specific, easily performed at low cost without the necessity of highly trained personnel or expensive laboratory equipment and which can be used, for example, for the detection of the AIDS virus.